Use of metal chelates in human or animal feeding

ABSTRACT

The present invention relates to the use in human and animal nutrition (monogastric and polygastric animals) of known chelates of bivalent metal Mg, Ca, Mn, Co, Cu, Zn and Fe with methionine hydroxy analogue. The present invention further relates to a method for preparing new chelates with methionine hydroxy analogue, both in solid form with iron (II), vanadium (iV) and (V) and molybdenum (V) and (VI), and in liquid form in aqueous solution with iron (II) and (III) and chrome (III). Eventually, the present invention relates to the use of said new chelates, both in solid form with iron (II), vanadium (IV) and (V) and molybdenum (V) and (VI), and in liquid form in aqueous solution with iron (II) and (III) and chrome (III), in human and animal nutrition.

The present invention relates to the use in human and animal nutrition(monogastric and polygastric animals) of known chelates of bivalentmetals Mg, Ca, Mn, Co, Cu, Zn and Fe with methionine hydroxy analogue.The present invention further relates to a method for preparing newchelates with methionine hydroxy analogue, both in solid form with iron(II), vanadium (IV) and (V) and molybdenum (V) and (VI), and in liquidform in aqueous solution with iron (II) and (III) and chrome (III).Eventually, the present invention relates to the use of said newchelates, both in solid form with iron (II), vanadium (IV) and (V) andmolybdenum (V) and (VI), and in liquid form in aqueous solution withiron (II) and (III) and chrome (III), in human and animal nutrition.

It is known that a metal chelate is a compound originating from anorganic molecule (such as an amino acid or a peptide chain or analpha-keto acid or an alpha-hydroxy acid) and a metal ion through strongcoordination bonds. Some known metal chelates are used in the field ofhuman nutrition. Their use is due to the biological action performed bythe metal element involved, as activator in several enzymatic reactions,and as regulator in various metabolic functions in all living organisms.The presence of a chelating molecule promotes the absorption,availability and use of the metal element since the latter is carried bythe organic component in all areas of the organism. This results also ina strong reduction of losses of unused metals in dejections, andtherefore in a significant economic saving and in an environmentaladvantage.

The general features that should characterize a highly bioavailablemetal chelate are: (a) neutrality of complex (the positive charge ofmetal is balanced by the negative charge of ligands; (b) absence ofnegative counter-ions (chlorides, sulfates); (c) low molecular weight ofcomplex (<1,500 Daltons); (d) well defined metal/chelating agent(bidentate) ratio, possibly ≦1:2.

Moreover, metal chelates should be obtained from simple and cleanprocesses with high yields starting from raw materials that can beeasily found. These features can be implemented by means of suitableligands, which should be easy to deproton and have at least two donoratoms in such a position to carry out chelation. Examples of ligands areamino acids and other organic acids suitably functionalized.

As far as iron is concerned, its administration in chelated form isparticularly efficient against anemia. Anemia is a pathologic status ofblood due to the reduction of the number of red blood cells, to thedecrease of the amount of hemoglobin or to both deficiencies. Iron isfundamental for hemoglobin since it is the center in which oxygen isfixed into heme. Classes of people who need a higher amount of iron aremenstruated or pregnant women, children under two years of age,vegetarians, people suffering from hemorrhoids, people suffering fromulcers and eventually blood donors. Symptoms felt by an anemic personare weariness, a higher sensibility to cold, irritableness, loss ofconcentration and heart palpitations.

Furthermore, iron can protect from viral or bacterial infections sinceit promotes the stimulation of the immune system. In addition, ironpromotes the metabolism of vitamins belonging to B group.

It is known that the lack of these vitamins can result in diseases suchas dermatitis or even more serious diseases such as pellagra (due to thedeficiency of vitamin B₃). Iron intervenes in processes of synthesis ofadrenaline and noradrenaline. Eventually, the lack of iron leads to aslow cicatrisation of wounds.

A first aim of the present invention is to suggest an integrator foradministration in human nutrition. Said integrator is also administeredto patients suffering from a deficiency of metal oligoelements such as:Mg, Ca, Mn, Co, Cu, Zn and Fe.

A second aim of the present invention is to suggest an integrator foragro-zootechnical nutrition to be administered to monogastric orpolygastric animals. Said integrator is also administered to monogastricor polygastric animals needing an administration of metal oligoelementssuch as: Mg, Ca, Mn, Co, Cu, Zn and Fe with a higher bioavailability.

A third aim of the present invention is to suggest a method forpreparing metal chelates with methionine hydroxy analogue or one of itssalts, both in solid form with iron (II), vanadium (IV) and/or vanadium(V) and molybdenum (V) and/or molybdenum (VI), and in liquid form inaqueous solution with iron (II) and (III) and chrome (III), in whichsaid metals are bonded to a bifunctional organic ligand with a strongcoordination bond so as to form a stable metal chelate.

A further aim of the present invention is to suggest the use of said newchelates, both in solid form with iron (II), vanadium (IV) and/orvanadium (V) and molybdenum (V) and/or molybdenum (VI), and in liquidform in aqueous solution with iron (II) and (III) and chrome (III), forpreparing metal integrators for human and animal nutrition.

In an embodiment of the present invention said first and second aim areachieved by suggesting the use of metal chelates [2:1] having thegeneral formula (I):(CH₃SCH₂CH₂CH(OH)COO)₂M.nH₂O (I)in which the bifunctional chelating agent is2-hydroxy-4-methylthiobutanoic acid, an alpha-hydroxy acid, known as“methionine hydroxy analogue” (MHA); M is a bivalent metal cation chosenfrom the group comprising: Co, Ca, Mg, Zn, Fe, Cu and Mn, and n is thenumber of water molecules; for preparing metal integrators for treatingpatients suffering from a deficiency of metal oligoelements or foradministration in the agro-zootechnical field to monogastric orpolygastric animals. In formula (I) there are zero to twelve watermolecules, preferably zero to six. For instance, zero to four.2-hydroxy-4-methylthiobutanoic acid builds with iron ion a chelatehaving a well defined stoichiometry, containing two molecules ofchelating agent pro iron atom, in the same way as it builds chelateswith bivalent metals Mg, Ca, Mn, Co, Cu and Zn.

The method for preparing metal chelates having formula (I) was describedin international patent application PCT/IT99/00225, and consists in thedirect reaction of MHA with the corresponding carbonates of bivalentmetals Mg, Ca, Mn Co, Cu and Zn.

In an embodiment according to the present invention the Applicant hasimproved a method for preparing metal chelates having formula (I), whichenvisages the direct reaction of MHA and metals (II) oxides such as: Mg,Ca, Mn, Co, Cu, Zn and Fe.

In this new embodiment oxides of metals (II) are used instead ofcarbonates as described in application PCT/IT99/00225, and technical andoperating conditions are unchanged with respect to those referred to inapplication PCT/IT99/00225, and therefore said operating conditions areregarded as contained in the present application.

Advantageously, the following are used: magnesium oxide mixed withmagnesium carbonate or alternatively completely replacing magnesiumcarbonate, zinc oxide mixed with zinc carbonate or alternativelycompletely replacing zinc carbonate, and calcium oxide mixed withcalcium carbonate or alternatively completely replacing calciumcarbonate.

The Applicant has found it advantageous to use for human nutrition thefollowing metal chelates:

-   (CH₃SCH₂CH₂CH(OH)COO)₂Zn.2H₂O

The two water molecules are not bonded to the metal.

-   (CH₃SCH₂CH₂CH(OH)COO)₂Cu

It is an anhydrous complex without water molecules bonded to the metal.

-   (CH₃SCH₂CH₂CH(OH)COO)₂Co.2H₂O

The two water molecules are complexed.

-   (CH₃SCH₂CH₂CH(OH)COO)₂Mn.2H₂O

The two water molecules are complexed.

-   (CH₃SCH₂CH₂CH(OH)COO)₂Ca.2H₂O

The two water molecules are not bonded to the metal.

-   (CH₃SCH₂CH₂CH(OH)COO)₂Mg.2H₂O

The two water molecules are not bonded to the metal.

In another embodiment of the present invention said third aim isachieved by suggesting a method for preparing metal chelates withmethionine hydroxy analogue or one of its salts, both in solid form withiron (II), vanadium (IV) and/or vanadium (V) and molybdenum (V) and/ormolybdenum (VI), and in liquid form in aqueous solution with iron (II)and (III) and chrome (III).

In the case of vanadium and molybdenum the method for preparing theirchelates consists in the reaction of their corresponding oxides withmethionine hydroxy analogue.

Alternatively, in the case of vanadium and molybdenum the method forpreparing their chelates consists in the reaction of their correspondingmetal salts with methionine hydroxy analogue.

For instance, an amount of solid V₂O₅ or MoO₃ is added with a solutionof methionine hydroxy analogue at a high temperature and under stirring.A transparent solution and a solid precipitate of a vanadium ormolybdenum chelate are obtained from the reaction.

In a preferred embodiment the Applicant has arranged in a receptaclevanadium oxide V₂O₅ and methionine hydroxy analogue MHA. Vanadium oxideand methionine hydroxy analogue are present in a molar ratio of 1:2 to1:8, preferably 1:4 to 1:6 (Vanadium/MHA).

The receptacle is equipped with stirring means, heating means and meansfor reaction reflux. The reaction takes placed under mechanical stirringand under reflux for a time between 20 and 60 minutes, preferably 30minutes. At the end of the reaction a dark green solution is obtained,from which after cooling a solid green precipitate is obtained. From thechemical and physical analysis of the precipitate the Applicant hasfound confirmation that it is a vanadium (IV) complex having formulaVOL₂, where L=deprotoned methionine hydroxy analogue.

During the reaction vanadium (V) oxide reduces to vanadium (IV).

In the case of iron (II) the method of preparation takes place byreaction of sodium salt (or an alkali metal or alkaline-earth metalsalt) of MHA with ferrous sulfate (or any other soluble iron (II) salt)in water environment.

The molar ratio MHA/Iron (II) is of 2:1 for completing the reaction.Ferrous chelate precipitates from the reaction environment and isfiltered and washed with water so as to eliminate soluble sodium sulfatewhich builds up.

After filtration and washing the chelated product is then dried so as toreduce the amount of absorbed water. The product is a pale yellowpowder, little water soluble, having formula:(CH₃SCH₂CH₂CH(OH)COO)₂Fe.2H₂O.

Water molecules are bonded directly to iron (TGA data).

The infrared vibrational spectrum shows the occurred chelation: as amatter of fact, the spectrum has a series of characteristic bandsaccording to the above structure.

In particular, the band due to the asymmetric stretching of carboxylicgroup can be observed at 1,596 cm⁻¹ significantly moved to lowfrequencies with respect to free MHA (1,720 cm⁻¹) as expected fordeprotonation and coordination. A pure and stable product is thusobtained.

In an embodiment of the present invention stable solutions of iron (II)and chrome (III) chelates with MHA can be obtained by dissolution inwater environment of soluble salts of iron or chrome (III) and of MHA ina ration MHA/M(III)≧2, preferably 3, and keeping pH at a suitable valueso as to prevent precipitation of the corresponding hydroxides.

In a preferred embodiment the Applicant has improved the preparation ofa stable solution of chrome (III) in which a chrome salt, for instancechrome sulfate Cr₂(SO₄)₃ is reacted with methionine hydroxy analogue MHAunder stirring, for instance by heating, for a time between 20 and 120minutes, preferably between 30 and 60 minutes. Chrome salt andmethionine hydroxy analogue are present in a molar ration of 1:2 to1:30, preferably 1:2 to 1:20. At the end of the reaction a solution ofchrome(III) complexes with methionine hydroxy analogue is obtained.

In another preferred embodiment of the present invention said fourth aimis achieved by suggesting the use of said new chelates, both in solidform with iron (II), vanadium (IV) and/or vanadium (V) and molybdenum(V) and/or molybdenum (VI), and in liquid form in aqueous solution withiron (II) and (III) and chrome (III), for the preparation of metalintegrators for human and animal nutrition.

Metal chelates described in the present invention can be used mixed onewith the other in various quantitative ratios for preparing metalintegrators. Therefore, the integrators suggested both for human andanimal nutrition can contain one or more metal chelates according to thepresent invention.

With reference to the metal chelates described above and according tothe present invention the Applicant has made a series of experimentaltests.

In Vitro Tests

For in vitro tests cells of human colon adenocarcinoma (CACO-2) havebeen used, these being the most frequently used in vitro system forstudies on intestinal functionality, in particular as far astransepithelial transport is concerned, since said cells (CACO-2)develop ultra-structural, functional and electrical properties that aresimilar to those of small intestine.

The formation of intercellular links can be monitored throughmeasurements of transepithelial electric resistance (TEER) of the singlelayer of cells. Since intercellular links limit the (para-cellular)movement of solutes, TEER alterations are commonly used as permeabilityindex of said links.

The equipment used for cell growth and differentiation is shown in FIG.1.

Said cells, referred to with A in FIG. 1, have been grown anddifferentiated on a carrier (permeable filter), referred to with B inFIG. 1, until a single layer of differentiated cells joined byfunctional intercellular links is formed. Said filter B separates theapical environment C, (AP), (simulating intestine lumen) from thebasolateral environment D, (BL), placed in the lower chamber, simulatingcapillary blood flow.

Said cells have been treated for 3 hours with two differentconcentrations of Fe(III)/MHA (1:3) and Fe(III)/NTA (1:2)(nitriltriacetic acid taken as reference chelate), in a buffer solutionat pH 5.5 and 37° C. Said solutions have been placed in the apicalcompartment C (AP), whereas the basolateral compartment D (BL) containedan iron-free solution of apotransferrin placed in a buffer solution atpH 7.4.

During said experiment, at pre-established time intervals, TEER has beenmeasured in Ω·cm². FIG. 2 (every 30 minutes over 3 hours) and FIG. 3after 24 hours.

The results are shown in FIG. 4, indicating the content of intracellulariron after 3 hours of treatment with Fe(III)/MHA and Fe(III)/NTA atdifferent concentrations. Data are expressed in nmoles iron/filter.

As can be inferred from FIG. 4, the passage of iron/MHA chelate from theapical environment, C, to the cell is higher than the one observed inthe control.

Moreover, from FIG. 5 (showing iron transport from apical environment Cto basolateral environment D after treatment with two differentconcentrations of Fe(III)/MHA and Fe(III)/NTA) it can be inferred thatthe concentration of transported iron is comparable. Data are expressedin nmoles iron/filter.

Data shown in FIGS. 4 and 5 confirm that iron chelate is stronglyabsorbed by cells of intestinal microvilli and moves within blood flow.

From FIG. 2 (showing TEER measurements) it can be inferred thatintercellular links are unchanged, thus proving the non-toxicity of ironchelate towards cells, contrarily to what happens in the case ofunchelated iron such as ferrous sulfate.

FIG. 3 shows the measurement of TEER 24 hours after the buffer solutionat 5.5 containing iron/MHA or iron/NTA has been removed keeping thecells in culture. Said FIG. 3 shows how iron chelate/MHA is stablewithin cells causing no toxic effect.

Finally, the tests show that MHA/M chelates according to the presentinvention are efficiently absorbed, stable within intestinal cells andnon-toxic.

The results shown above support the use of said new chelates, both insolid form with iron (II), vanadium (IV) and/or vanadium (V) andmolybdenum (V) and/or molybdenum (VI), and in liquid form in aqueoussolution with iron (II) and (III) and chrome (III), for the preparationof metal integrators for human and animal nutrition.

In Vivo Tests

Said tests have involved both monogastric animals (such as pigs) andpolygastric animals (such as calves).

a) Monogastric Animals (Pigs)

Two test groups of pigs (Control and Test) of 35 days of age and weanedat 19 days, were administered a food differing only in the zinc source.

Said food consisted, pro kg of food as such, of 3,500 Kcal ED, 1.15 g oflysine, and a total amount of zinc element of 81 mg (81 ppm), of which31 mg (31 ppm) were given by raw materials, whereas 50 mg (50 ppm) wererespectively in form of zinc sulfate (Control) and zinc chelate with MHA(Test) according to the present invention.

The two groups of animals were balanced by nest, living weight and sexand were fed for 27 days. Other 4 animals were immediately sacrificedand a sampling was made of them according to the following procedure.The two groups of animals were weighed before starting the test andafter 27 days. At the end of the test the pigs were sacrificed andstomach, intestine, left kidney and liver were removed from every pig.Stomach and intestine were emptied and weighed again so as to obtain thenet weight of said organs. A sample of kidney, liver and brain was takenand frozen. Stomach, intestine, left kidney and liver were then puttogether with the remaining carcass and homogenized with a mixer. Asample was then taken from the mass thus obtained and frozen. Thesamples were then dehydrated with a dehydrating device and underwentchemical analysis.

The level of zinc, copper and iron were determined on the lyophilizedsamples by atomic absorption spectroscopy. The body levels of zinc werereferred to the product.

On the basis of the body's “net” weight (i.e. without the content of thedigestive tube) and of its content of zinc at test beginning and end itwas possible to determine also daily zinc retention. The results of thechemical analyses carried out on the samples are shown in Table 1. Thepigs were sacrificed at an average weight of 16.2 kg. The average dailyweight increase was of 324 g.

As can be inferred from the data shown in Table 2 concerning daily zincretention of the two different sources, the integration of zinc chelatewas retained by the organism 26% more (P=0.07) than the integration withzinc sulfate. Table 3 shows the data concerning the effect of the zincsource on the content of zinc, copper and iron in liver, kidney andbrain and, therefore, on the interaction with said elements present inthe diet under inorganic form. As a matter of fact, it is known aboutthe interaction exerted by said free ions by reducing one the absorptionof the other.

The content of said three minerals in liver was not affected by the dietand therefore by the zinc source. Average values were 296 mg/kg forzinc, 63 mg/kg for copper and 220 for iron. Conversely, kidney showed ahigher content of zinc (+18%, P=0.07), of iron (+36%, P=0.01) and ofcopper (+36%, P=0.12), and did not reach only for the latter value thethreshold of statistic significance, though it showed a tendency towardsan increase in the retention of said metal element.

In brain there was a tendency towards a higher content of zinc (+13%),of copper (+20%), of iron (+25%).

The obtained results point out a higher bioavailability of the metalelement in chelated form with respect to inorganic sources such assulfates, and further a lower interaction with other ions, which resultsin a higher retention of the latter.

As is known, pigs are one of man-closest animal models and as such theyare often used as model for evaluations and studies in the human field.

b) Polygastric Animals (Meat Cattle)

Two groups of female Charolaise calves (30 months old) comprising 6animals each, with an average starting weight of 567 kg (Control and of565 kg (Test), were fed for 90 days with the same diet. The onlydifference was that the Control group was administered zinc carbonateand the Test group zinc chelate according to the present invention.Daily ingestion was of 22 kg/animal and the total daily supply of zincelement was of 700 mg.

Living weight at test beginning and end, dead weight and slaughteringyield were determined for each animal. Data are shown in Table 4.

Animals fed with zinc chelate with respect to those fed with zinccarbonate have a significantly higher final weight (652 kg vs. 642 kg,p<0.05), a significantly higher daily weight increase (1,039 g vs. 934g, p<0.05), a significantly higher carcass weight (377 kg vs. 366 kg,P<0.01), and a significantly higher yield (57.83% vs. 57.03%, p<0.01).Data are shown in Table 4. The effect due to the presence of zincchelate in the ratio administered to animals is shown in Table 5.

Said results show an evident improvement of the aforesaid zootechnicalperformances of zinc chelate with respect to inorganic sources of saidelement.

Stability constants for the various Fe/MHA complexes have beencalculated with potentiometric titrations. The stability of iron (III)complexes is very high and chelated species form also at acid pH.Uncomplexed Fe³⁺ ions are present only at very low pH values (<2.5),whereas at higher pH values all iron (III) is complexed as chelatedspecies metal/ligand=1:2. TABLE NO. 1 Chelate Sulfate E.M.S. Number ofdays n 26.3 26.4 Initial living weight kg 7.54 7.33 0.50 Final livingweight kg 16.14 16.20 0.55 Daily increase g 327 321 11 Diet g 544 5481.9 Conversion index 1.68 1.72 0.054 Initial net living weight kg 7.317.49 0.48 Final net weight kg 15.57 15.76 0.55 Daily net increase g 314313 11

TABLE NO. 2 Chelate Sulfate E.M.S. Daily Zn retention mg 5.40 5.27 0.37

TABLE NO. 3 Statistic Chelate Sulfate E.M.S. significance Liver Zinc 292300 21 P = 0.80 Copper 63.6 62.5 6.3 P = 0.91 Iron 240 199 35 P = 0.44Kidney Zinc 97.5 82.3 5.5 P = 0.07 Copper 55.0 40.2 6.4 P = 0.12 Iron244 179 11.8 P < 0.01 Brain Zinc 53.9 47.6 3.5 P = 0.23 Copper 28.1 23.33.5 P = 0.35 Iron 100.9 80.6 9.9 P = 0.17

TABLE NO. 4 Diet Initial weight E.M.S. Control 567.700000 31.845747 Zn565.633333 31.845747 Final weight E.M.S. Control 642. 2.694781 Zn 652.2.691923 Control vs. Zn P < 0.05 Diet Daily increase E.M.S. Co 931.29.94201 Zn 1,039. 29.91025 Control vs. Zn P < 0.05 Diet Carcass weightE.M.S. Control 366. 1.812368 Zn 377. 1.810445 Control vs. Zn P < 0.01Slaughtering Diet weight E.M.S. Control 57.03 0.1132212 Zn 57.850.1131011 Control vs. Zn P < 0.01

TABLE NO. 5 Initial Final Total Dead Slaughtering Calf weight weightDaily increase weight weight number kg kg increase g kg kg kg CONTROL 1583.5 670.75 1,050 94.50 383 0.578 2 583.5 670.75 970 87.30 383 0.571 3599.5 675.96 850 76.50 388 0.574 4 540.9 623.67 920 82.80 353 0.566 5568.1 649.12 900 81.00 370 0.570 6 532.3 624.13 1,020 91.80 357 0.572TEST 1 625.6 724.61 1,100 99.0 421 0.581 2 676.8 789.29 1,250 112.5 4570.579 3 582.7 686.21 1,150 103.5 398 0.580 4 564.0 653.98 1,000 90.00378 0.578 5 476.5 557.49 900 81.00 320 0.574 6 468.2 548.28 890 80.10318 0.580

1-25. (canceled)
 26. A method for preparing an integrator comprising atleast one metal chelate selected from the metal chelate of generalformula (I):(CH₃SCH₂CH₂CH(OH)COO)₂M.nH₂O  (I) wherein: M is a bivalent metal cationselected from the group comprising of: Mg, Ca, Mn, Co, Cu, Zn and Fe; nis the number of water molecules.
 27. The method according to claim 26,wherein said at least one metal chelate is selected from the groupconsisting of: (CH₃SCH₂CH₂CH(OH)COO)₂Zn.2H₂O; (CH₃SCH₂CH₂CH(OH)COO)₂Cu;(CH₃SCH₂CH₂CH(OH)COO)₂Co.2H₂O; (CH₃SCH₂CH₂CH(OH)COO)₂Mn.2H₂O;(CH₃SCH₂CH₂CH(OH)COO)₂Ca.2H₂O; (CH₃SCH₂CH₂CH(OH)COO)₂Mg.2H₂O; or(CH₃SCH₂CH₂CH(OH)COO)₂Fe.2H₂O.
 28. The method according to claim 27,wherein the integrator is administered to human beings or animalssuffering from a deficiency of metal oligoelements such as Mg, Ca, Mn,Co, Cu, Zn and Fe.
 29. The method according to claim 28, wherein theintegrator is administered to monogastric or polygastric animals.
 30. Amethod for preparing a metal chelate of formulaCH₃SCH₂CH₂CH(OH)COO)₂Fe.2H₂O comprising a step in which an alkali metalor alkaline-earth metal salt of methionine hydroxy analogue is reactedwith a soluble iron (II) salt in water.
 31. The method according toclaim 30, wherein said alkali metal salt is a sodium salt of methioninehydroxy analogue and said soluble iron (II) salt is a ferrous sulfate.32. The method according to claim 31, wherein said iron (II) chelateobtained from the reaction is filtered and washed with water.
 33. Acomposition comprising water and at least one complex of general formula[Methionine Hydroxy Analogue:M(III)] wherein: M(III) is selected fromiron (III) or chrome (III) and said at least one complex has a molarratio between Methionine Hydroxy Analogue and M(III) equal or biggerthan
 2. 34. The composition according to claim 33, wherein the metalcomplex is [Methionine Hydroxy Analogue:Fe(III)].
 35. The compositionaccording to claim 33, wherein the metal complex is [Methionine HydroxyAnalogue:Cr(III)].
 36. A method for preparing an integrator comprisingat least one complex according to claim
 33. 37. The method according toclaim 36, wherein the integrator is administered to human beings oranimals suffering from a deficiency of metal oligoelements such as Feand Cr.
 38. The method according to claim 37, wherein the integrator isadministered to human beings or monogastric or polygastric animals.